海洋曲霉属真菌次级代谢产物生物合成基因簇的基因组挖掘与遗传转化体系构建
发布时间：2018-12-12 17:33
【摘要】：海洋微生物来源的天然产物因其独特的结构与生物学活性而备受关注,特别是有研究证实海洋动植物体内的微生物是其活性化合物的真正生产者。海蟑螂(Ligiaoceanic)是中国南方沿海渔民治疗跌打损伤常用的药用动物,但目前对海蟑螂及其微生物的次级代谢产物(Secondary Metabolites,SMs)的研究较少,难以确认其药用活性成分。本研究以海蟑螂肠道内海洋真菌Z5为研究对象,经rDNA序列鉴定为曲霉属菌株(Aspergillussp.)。应用二代高通量测序技术对该菌株进行全基因组测序分析及antiSMASH基因簇分析,结果显示Z5菌株基因组大小为33.8 Mbp,系统进化处于A.versicolor分支,共含有89个SMs生合成基因簇,结合One-Strain-Many-Compounds(OSMAC)结果说明:该菌具有合成多种SMs的潜能;全基因组挖掘的SMs基因簇数量远超OSMAC分析的化合物数量,多数基因簇在现有培养条件下为沉默状态,如激活这些沉默基因簇的表达,极可能获得新型活性化合物。为开展该菌株的生物合成研究而获得新型化合物,本研究构建了 Z5的遗传转化系统,探索优化原生质体制备纯化方法,确定可通过β-巯基乙醇预处理及蔗糖分层纯化法获得再生率为94%的高质量原生质体,并应用Double-Joint PCR替代传统酶切连接法构建基因盒,最终通过PEG/CaCl2转化,获得Z5的营养缺陷型菌株。本研究为海洋真菌新型天然产物的获得提供重要来源,为开发应用该类真菌天然产物奠定基础,有助于阐明海洋真菌系统进化及与海洋动植物活性产物的关联,为开发利用其它海洋真菌活性天然产物提供经验方法。
[Abstract]:The natural products derived from marine microorganisms have attracted much attention because of their unique structure and biological activities. Especially, some studies have confirmed that the microorganisms in marine animals and plants are the real producers of their active compounds. Sea cockroach (Ligiaoceanic) is a common medicinal animal used by fishermen in southern China to treat the damage. However, there are few studies on the secondary metabolites (Secondary Metabolites,SMs) of sea cockroaches and their microbes, so it is difficult to confirm the medicinal active components of sea cockroaches. In this study, the marine fungus Z5 in the intestinal tract of sea cockroach was identified as (Aspergillussp.). By rDNA sequence analysis. The second generation high-throughput sequencing technique was used to analyze the whole genome and the antiSMASH gene cluster of the strain. The results showed that the genome size of Z5 strain was 33.8 Mbp, phylogeny in the A.versicolor branch, and there were 89 SMs synthetic gene clusters. The results of One-Strain-Many-Compounds (OSMAC) showed that the strain had the potential to synthesize multiple SMs. The number of SMs gene clusters mined by the whole genome is much larger than the number of compounds analyzed by OSMAC. Most of the gene clusters are silenced under the existing culture conditions. If the expression of these silent gene clusters is activated, it is very possible to obtain new active compounds. In order to study the biosynthesis of the strain and obtain new compounds, the genetic transformation system of Z5 was constructed to optimize the preparation and purification of protoplasts. High quality protoplasts with regeneration rate of 94% could be obtained by 尾 -mercaptoethanol pretreatment and sucrose stratification purification. The gene cassette was constructed by Double-Joint PCR instead of traditional enzyme ligation method, and finally transformed by PEG/CaCl2. A nutrient deficient strain of Z5 was obtained. This study provides an important source for the acquisition of new natural products of marine fungi, lays a foundation for the development and application of these natural products, and helps to clarify the phylogeny of marine fungi and their association with the active products of marine flora and fauna. To provide an empirical method for the development and utilization of other marine fungi active natural products.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933
本文编号：2375003
[Abstract]:The natural products derived from marine microorganisms have attracted much attention because of their unique structure and biological activities. Especially, some studies have confirmed that the microorganisms in marine animals and plants are the real producers of their active compounds. Sea cockroach (Ligiaoceanic) is a common medicinal animal used by fishermen in southern China to treat the damage. However, there are few studies on the secondary metabolites (Secondary Metabolites,SMs) of sea cockroaches and their microbes, so it is difficult to confirm the medicinal active components of sea cockroaches. In this study, the marine fungus Z5 in the intestinal tract of sea cockroach was identified as (Aspergillussp.). By rDNA sequence analysis. The second generation high-throughput sequencing technique was used to analyze the whole genome and the antiSMASH gene cluster of the strain. The results showed that the genome size of Z5 strain was 33.8 Mbp, phylogeny in the A.versicolor branch, and there were 89 SMs synthetic gene clusters. The results of One-Strain-Many-Compounds (OSMAC) showed that the strain had the potential to synthesize multiple SMs. The number of SMs gene clusters mined by the whole genome is much larger than the number of compounds analyzed by OSMAC. Most of the gene clusters are silenced under the existing culture conditions. If the expression of these silent gene clusters is activated, it is very possible to obtain new active compounds. In order to study the biosynthesis of the strain and obtain new compounds, the genetic transformation system of Z5 was constructed to optimize the preparation and purification of protoplasts. High quality protoplasts with regeneration rate of 94% could be obtained by 尾 -mercaptoethanol pretreatment and sucrose stratification purification. The gene cassette was constructed by Double-Joint PCR instead of traditional enzyme ligation method, and finally transformed by PEG/CaCl2. A nutrient deficient strain of Z5 was obtained. This study provides an important source for the acquisition of new natural products of marine fungi, lays a foundation for the development and application of these natural products, and helps to clarify the phylogeny of marine fungi and their association with the active products of marine flora and fauna. To provide an empirical method for the development and utilization of other marine fungi active natural products.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933
【参考文献】
相关期刊论文 前6条
1 朱伟明;王俊锋;;海洋真菌生物活性物质研究之管见[J];菌物学报;2011年02期
2 郭跃伟;;海洋天然产物和海洋药物研究的历史、现状和未来[J];自然杂志;2009年01期
3 ;Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation[J];Chemical Research in Chinese Universities;2008年05期
4 许静;徐俊;;海洋共附生微生物天然产物生物合成基因研究进展[J];微生物学报;2008年07期
5 孙剑秋,于寒颖,张鹏,张彦丰,凌洪博,解玉红,周东坡,平文祥;树状多节孢原生质体的制备和再生[J];应用与环境生物学报;2001年04期
6 汪天虹,肖天,朱汇源,汪浩;海洋丝状真菌生物活性物质研究进展[J];海洋科学;2001年06期
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